Project Summary/Abstract This application is to continue studies on the cross-talk between the transcription factors RUNX1 and GATA-1 in megakaryocytic differentiation. Transcriptional pathways that dictate megakaryocyte differentiation remain poorly characterized but will most likely provide important targets in the design of treatments for platelet production defects and for megakaryocytic leukemias. Biochemical, genetic, and clinical data all strongly support the coexistence of GATA-1 and RUNX1 in a common molecular pathway critical for normal megakaryocytic development. By contrast, the related factor GATA-2 lacks transcriptional cooperation with RUNX1 and cannot compensate for loss of GATA-1 in megakaryopoiesis in vivo. Functional mapping of GATA-1/GATA-2 chimeras has identified a 67 amino acid segment in the GATA-1 amino terminus critical for transcriptional cooperation with RUNX1. Mapping within RUNX1 has indicated requirements for DNA binding by the RUNT domain and for S/TP phosphoacceptor sites within the carboxy terminal region, particularly S400. Importantly, GATA-1 promotes hyperphosphorylation of RUNX1, and GATA-1/GATA-2 chimeras that lack transcriptional synergy with RUNX1 fail to induce RUNX1 hyperphosphorylation. Pharmacologic and molecular screens have identified Cdk9 as the kinase critical for transcriptional cooperation of RUNX1 and GATA-1. Indeed, GATA-1 recruits Cdk9 to RUNX1 transcriptional complexes, promoting phosphorylation of RUNX1 and associated factors. Interestingly, GATA-2 also binds Cdk9 but induces the downregulation of the Cdk9 p55 isoform. Cdk9 has been shown to participate in transcriptional elongation, cellular hypertrophy, and induction of RUNX1 expression during zebrafish hematopoietic ontogeny. In our unpublished data, inhibition of Cdk9 causes blockade of primary human megakaryocytic differentiation. In our mouse model, a synthetic lethal interaction exists between Cdk9 inhibition and GATA-1 deficiency. In particular, the GATA-1Lo strain, with a megakaryocte-specific deficit in GATA-1 expression, uniquely responds to Cdk9 inhibition by developing a fulminant but reversible megakaryoblastic proliferative disorder. This distinctive phenotype recalls a reversible human disease, the transient megakaryocytic proliferative disorder of Down syndrome, associated with somatic mutations of GATA-1. We also provide in vivo genetic evidence for GATA-1-RUNX1 megakaryocytic cooperation: deletion in mice of a single RUNX1 allele on a GATA-1Lo background causes spontaneous muscle bleeds and marrow dysmegakaryopoiesis. We therefore postulate: a) that GATA-1 promotes the recruitment of active Cdk9 to RUNX1 complexes, leading to RUNX1 activation, enhanced transcriptional elongation of megakaryocyte genes and consequent acquisition of a [unreadable]hypertrophic[unreadable] megakaryocyte phenotype, b) that GATA-2 fails to recruit active Cdk9 because of its induction of Cdk9 p55 downregulation, and c) that in vivo defects in this pathway contribute to megakaryocyte proliferative disorders and dysmegakaryopoiesis. Aim I will test this model through a molecular dissection of GATA-1 mediated Cdk9 signaling to RUNX1. Aim II will further explore the model in murine model systems of megakaryopoiesis, employing GATA-1 and RUNX1 knockout strains.